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B-cell lymphoma 2-related protein A1 (BCL2A1) is a member of the BCL-2 family of anti-apoptotic proteins that confers resistance to treatment with anti-cancer drugs; however, there are presently no agents that target BCL2A1. The MUC1-C oncoprotein is aberrantly expressed in triple-negative breast cancer (TNBC) cells, induces the epithelial-mesenchymal transition (EMT) and promotes anti-cancer drug resistance. The present study demonstrates that targeting MUC1-C genetically and pharmacologically in TNBC cells results in the downregulation of BCL2A1 expression. The results show that MUC1-C activates the BCL2A1 gene by an NF-κB p65-mediated mechanism, linking this pathway with the induction of EMT. The MCL-1 anti-apoptotic protein is also of importance for the survival of TNBC cells and is an attractive target for drug development. We found that inhibiting MCL-1 with the highly specific MS1 peptide results in the activation of the MUC1-C→NF-κB→BCL2A1 pathway. In addition, selection of TNBC cells for resistance to ABT-737, which inhibits BCL-2, BCL-xL and BCL-W but not MCL-1 or BCL2A1, is associated with the upregulation of MUC1-C and BCL2A1 expression. Targeting MUC1-C in ABT-737-resistant TNBC cells suppresses BCL2A1 and induces death, which is of potential therapeutic importance. These findings indicate that MUC1-C is a target for the treatment of TNBCs unresponsive to agents that inhibit anti-apoptotic members of the BCL-2 family., Competing Interests: D.K. has equity interests in, serves as a member of the board of directors of and is a paid consultant to Genus Oncology. The remaining authors declare no competing interests.

The immune checkpoint ligand PD-L1 and the transmembrane mucin MUC1 are upregulated in triple-negative breast cancer (TNBC), where they contribute to its aggressive pathogenesis. Here, we report that genetic or pharmacological targeting of the oncogenic MUC1 subunit MUC1-C is sufficient to suppress PD-L1 expression in TNBC cells. Mechanistic investigations showed that MUC1-C acted to elevate PD-L1 transcription by recruitment of MYC and NF-κB p65 to the PD-L1 promoter. In an immunocompetent model of TNBC in which Eo771/MUC1-C cells were engrafted into MUC1 transgenic mice, we showed that targeting MUC1-C associated with PD-L1 suppression, increases in tumor-infiltrating CD8 + T cells and tumor cell killing. MUC1 expression in TNBCs also correlated inversely with CD8, CD69, and GZMB, and downregulation of these markers associated with decreased survival. Taken together, our findings show how MUC1 contributes to immune escape in TNBC, and they offer a rationale to target MUC1-C as a novel immunotherapeutic approach for TNBC treatment. Significance: These findings show how upregulation of the transmembrane mucin MUC1 contributes to immune escape in an aggressive form of breast cancer, with potential implications for a novel immunotherapeutic approach. Cancer Res; 78(1); 205-15. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The polycomb repressive complex 1 (PRC1) includes the BMI1, RING1 and RING2 proteins. BMI1 is required for survival of multiple myeloma (MM) cells. The MUC1-C oncoprotein is aberrantly expressed by MM cells, activates MYC and is also necessary for MM cell survival. The present studies show that targeting MUC1-C with (i) stable and inducible silencing and CRISPR/Cas9 editing and (ii) the pharmacologic inhibitor GO-203, which blocks MUC1-C function, downregulates BMI1, RING1 and RING2 expression. The results demonstrate that MUC1-C drives BMI1 transcription by a MYC-dependent mechanism. MUC1-C thus promotes MYC occupancy on the BMI1 promoter and thereby activates BMI1 expression. We also show that the MUC1-C→MYC pathway induces RING2 expression. Moreover, in contrast to BMI1 and RING2, we found that MUC1-C drives RING1 by an NF-κB p65-dependent mechanism. Targeting MUC1-C and thereby the suppression of these key PRC1 proteins was associated with downregulation of the PRC1 E3 ligase activity as evidenced by decreases in ubiquitylation of histone H2A. Targeting MUC1-C also resulted in activation of the PRC1-repressed tumor suppressor genes, PTEN, CDNK2A and BIM . These findings identify a heretofore unrecognized role for MUC1-C in the epigenetic regulation of MM cells., Competing Interests: CONFLICTS OF INTEREST DK has ownership interest (including patents) and is a consultant/advisory board member of Genus Oncology. No potential conflicts of interest were disclosed by the other authors.

The EZH2 histone methyltransferase is a member of the polycomb repressive complex 2 (PRC2) that is highly expressed in diverse human cancers and is associated with a poor prognosis. MUC1-C is an oncoprotein that is similarly overexpressed in carcinomas and has been linked to epigenetic regulation. A role for MUC1-C in regulating EZH2 and histone methylation is not known. Here, we demonstrate that targeting MUC1-C in diverse human carcinoma cells downregulates EZH2 and other PRC2 components. MUC1-C activates (i) the EZH2 promoter through induction of the pRB→E2F pathway, and (ii) an NF-κB p65 driven enhancer in exon 1. We also show that MUC1-C binds directly to the EZH2 CXC region adjacent to the catalytic SET domain and associates with EZH2 on the CDH1 and BRCA1 promoters. In concert with these results, targeting MUC1-C downregulates EZH2 function as evidenced by (i) global and promoter-specific decreases in H3K27 trimethylation (H3K27me3), and (ii) activation of tumor suppressor genes, including BRCA1. These findings highlight a previously unreported role for MUC1-C in activating EZH2 expression and function in cancer cells.

Immunotherapeutic approaches, particularly programmed death 1/programmed death ligand 1 (PD-1/PD-L1) blockade, have improved the treatment of non-small-cell lung cancer (NSCLC), supporting the premise that evasion of immune destruction is of importance for NSCLC progression. However, the signals responsible for upregulation of PD-L1 in NSCLC cells and whether they are integrated with the regulation of other immune-related genes are not known. Mucin 1 (MUC1) is aberrantly overexpressed in NSCLC, activates the nuclear factor-κB (NF-κB) p65→︀ZEB1 pathway and confers a poor prognosis. The present studies demonstrate that MUC1-C activates PD-L1 expression in NSCLC cells. We show that MUC1-C increases NF-κB p65 occupancy on the CD274/PD-L1 promoter and thereby drives CD274 transcription. Moreover, we demonstrate that MUC1-C-induced activation of NF-κB→︀ZEB1 signaling represses the TLR9 (toll-like receptor 9), IFNG, MCP-1 (monocyte chemoattractant protein-1) and GM-CSF genes, and that this signature is associated with decreases in overall survival. In concert with these results, targeting MUC1-C in NSCLC tumors suppresses PD-L1 and induces these effectors of innate and adaptive immunity. These findings support a previously unrecognized central role for MUC1-C in integrating PD-L1 activation with suppression of immune effectors and poor clinical outcome.

The cancer immune microenvironment is of importance for the effectiveness of immunotherapy; however, its dysregulation is poorly understood. The MUC1-C oncoprotein is aberrantly overexpressed in non-small cell lung cancer (NSCLC) and has been linked to the induction of PD-L1. The present work investigated the effects of targeting MUC1-C in an immuno-competent MUC1 transgenic (MUC1.Tg) mouse model. We show that Lewis Lung Carcinoma cells expressing MUC1-C (LLC/MUC1) exhibit upregulation of PD-L1 and suppression of interferon-γ (IFN-γ). In studies of LLC/MUC1 cells growing in vitro and as tumors in MUC1.Tg mice, treatment with the MUC1-C inhibitor, GO-203, was associated with the downregulation of PD-L1 and induction of IFN-γ. The results further demonstrate that targeting MUC1-C results in enhanced effector function of CD8+ tumor-infiltrating lymphocytes (TILs) as evidenced by increased expression of the activation marker CD69, the degranulation marker CD107α, and granzyme B. Notably, targeting MUC1-C was also associated with marked increases in TIL-mediated killing of LLC/MUC1 cells. Analysis of gene expression data sets further showed that overexpression of MUC1 in NSCLCs correlates negatively with CD8, IFNG and GZMB , and that decreases in CD8 and IFNG are associated with poor clinical outcomes. These findings in LLC/MUC1 tumors and in NSCLCs indicate that MUC1-C→PD-L1 signaling promotes the suppression of CD8+ T-cell activation and that MUC1-C is a potential target for reprogramming of the tumor microenvironment.

B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) is a component of the polycomb repressive complex 1 (PRC1) complex that is overexpressed in breast and other cancers, and promotes self-renewal of cancer stem-like cells. The oncogenic mucin 1 (MUC1) C-terminal (MUC1-C) subunit is similarly overexpressed in human carcinoma cells and has been linked to their self-renewal. There is no known relationship between MUC1-C and BMI1 in cancer. The present studies demonstrate that MUC1-C drives BMI1 transcription by a MYC-dependent mechanism in breast and other cancer cells. In addition, we show that MUC1-C blocks miR-200c-mediated downregulation of BMI1 expression. The functional significance of this MUC1-C→︀BMI1 pathway is supported by the demonstration that targeting MUC1-C suppresses BMI1-induced ubiquitylation of H2A and thereby derepresses homeobox HOXC5 and HOXC13 gene expression. Notably, our results further show that MUC1-C binds directly to BMI1 and promotes occupancy of BMI1 on the CDKN2A promoter. In concert with BMI1-induced repression of the p16 INK4a tumor suppressor, we found that targeting MUC1-C is associated with induction of p16 INK4a expression. In support of these results, analysis of three gene expresssion data sets demonstrated highly significant correlations between MUC1-C and BMI1 in breast cancers. These findings uncover a previously unrecognized role for MUC1-C in driving BMI1 expression and in directly interacting with this stem cell factor, linking MUC1-C with function of the PRC1 in epigenetic gene silencing.

Aberrant expression of the DNA methyltransferases (DNMTs) and disruption of DNA methylation patterns are associated with carcinogenesis and cancer cell survival. The oncogenic MUC1-C protein is aberrantly overexpressed in diverse carcinomas; however, there is no known link between MUC1-C and DNA methylation. Our results demonstrate that MUC1-C induces the expression of DNMT1 and DNMT3b, but not DNMT3a, in breast and other carcinoma cell types. We show that MUC1-C occupies the DNMT1 and DNMT3b promoters in complexes with NF-κB p65 and drives DNMT1 and DNMT3b transcription. In this way, MUC1-C controls global DNA methylation as determined by analysis of LINE-1 repeat elements. The results further demonstrate that targeting MUC1-C downregulates DNA methylation of the CDH1 tumor suppressor gene in association with induction of E-cadherin expression. These findings provide compelling evidence that MUC1-C is of functional importance to induction of DNMT1 and DNMT3b and, in turn, changes in DNA methylation patterns in cancer cells., Competing Interests: Author Information: The authors declare competing financial interests: D.K. holds equity in Genus Oncology and is a consultant to the company. The other authors disclosed no potential conflicts of interest.

Apical-basal polarity and epithelial integrity are maintained in part by the Crumbs (CRB) complex. The C--terminal subunit of MUC1 (MUC1-C) is a transmembrane protein that is expressed at the apical border of normal epithelial cells and aberrantly at high levels over the entire surface of their transformed counterparts. However, it is not known whether MUC1-C contributes to this loss of polarity that is characteristic of carcinoma cells. Here it is demonstrated that MUC1-C downregulates expression of the Crumbs complex CRB3 protein in triple-negative breast cancer (TNBC) cells. MUC1-C associates with ZEB1 on the CRB3 promoter and represses CRB3 transcription. Notably, CRB3 activates the core kinase cassette of the Hippo pathway, which includes LATS1 and LATS2. In this context, targeting MUC1-C was associated with increased phosphorylation of LATS1, consistent with activation of the Hippo pathway, which is critical for regulating cell contact, tissue repair, proliferation, and apoptosis. Also shown is that MUC1-C--mediated suppression of CRB3 and the Hippo pathway is associated with dephosphorylation and activation of the oncogenic YAP protein. In turn, MUC1-C interacts with YAP, promotes formation of YAP/β-catenin complexes, and induces the WNT target gene MYC. These data support a previously unrecognized pathway in which targeting MUC1-C in TNBC cells (i) induces CRB3 expression, (ii) activates the CRB3-driven Hippo pathway, (iii) inactivates YAP, and thereby (iv) suppresses YAP/β-catenin-mediated induction of MYC expression., Implications: These findings demonstrate a previously unrecognized role for the MUC1-C oncoprotein in the regulation of polarity and the Hippo pathway in breast cancer. Mol Cancer Res; 14(12); 1266-76. ©2016 AACR., Competing Interests: Potential Conflict of Interest: The authors declare competing financial interests: D.K. holds equity in Genus Oncology and is a consultant to the company. The other authors disclosed no potential conflicts of interest., (©2016 American Association for Cancer Research.)

Aberrant DNA methylation is a hallmark of acute myeloid leukemia (AML); however, the regulation of DNA methyltransferase 1 (DNMT1), which is responsible for maintenance of DNA methylation patterns, has largely remained elusive. MUC1-C is a transmembrane oncoprotein that is aberrantly expressed in AML stem-like cells. The present studies demonstrate that targeting MUC1-C with silencing or a pharmacologic inhibitor GO-203 suppresses DNMT1 expression. In addition, MUC1 expression positively correlates with that of DNMT1 in primary AML cells, particularly the CD34+/CD38- population. The mechanistic basis for this relationship is supported by the demonstration that MUC1-C activates the NF-κB p65 pathway, promotes occupancy of the MUC1-C/NF-κB complex on the DNMT1 promoter and drives DNMT1 transcription. We also show that targeting MUC1-C substantially reduces gene promoter-specific DNA methylation, and derepresses expression of tumor suppressor genes, including CDH1, PTEN and BRCA1. In support of these results, we demonstrate that combining GO-203 with the DNMT1 inhibitor decitabine is highly effective in reducing DNMT1 levels and decreasing AML cell survival. These findings indicate that (i) MUC1-C is an attractive target for the epigentic reprogramming of AML cells, and (ii) targeting MUC1-C in combination with decitabine is a potentially effective clinical approach for the treatment of AML., Competing Interests: D.K holds equity in Genus Oncology and is a consultant to the company. The remaining authors declare no conflict of interest.

Multiple myeloma (MM) cell lines and primary tumor cells are addicted to the MYC oncoprotein for survival. Little is known, however, about how MYC expression is upregulated in MM cells. The mucin 1 C-terminal subunit (MUC1-C) is an oncogenic transmembrane protein that is aberrantly expressed in MM cell lines and primary tumor samples. The present studies demonstrate that targeting MUC1-C with silencing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 editing or with the GO-203 inhibitor is associated with downregulation of MYC messenger RNA and protein. The results show that MUC1-C occupies the MYC promoter and thereby activates the MYC gene by a β-catenin/transcription factor 4 (TCF4)-mediated mechanism. In this way, MUC1-C (1) increases β-catenin occupancy on the MYC promoter, (2) forms a complex with β-catenin and TCF4, and, in turn, (3) drives MYC transcription. Analysis of MM cells using quantitative real-time reverse transcription polymerase chain reaction arrays further demonstrated that silencing MUC1-C is associated with downregulation of MYC target genes, including CCND2, hTERT, and GCLC Analysis of microarray data sets further demonstrated that MUC1 levels positively correlate with MYC expression in MM progression and in primary cells from over 800 MM patients. These findings collectively provide convincing evidence that MUC1-C drives MYC expression in MM., (© 2016 by The American Society of Hematology.)

Dysregulation of MYC expression is a hallmark of cancer, but the development of agents that target MYC has remained challenging. The oncogenic MUC1-C transmembrane protein is, like MYC, aberrantly expressed in diverse human cancers. The present studies demonstrate that MUC1-C induces MYC expression in KRAS mutant non-small cell lung cancer (NSCLC) cells, an effect that can be suppressed by targeting MUC1-C via shRNA silencing, CRISPR editing, or pharmacologic inhibition with GO-203. MUC1-C activated the WNT/β-catenin (CTNNB1) pathway and promoted occupancy of MUC1-C/β-catenin/TCF4 complexes on the MYC promoter. MUC1-C also promoted the recruitment of the p300 histone acetylase (EP300) and, in turn, induced histone H3 acetylation and activation of MYC gene transcription. We also show that targeting MUC1-C decreased the expression of key MYC target genes essential for the growth and survival of NSCLC cells, such as TERT and CDK4. Based on these results, we found that the combination of GO-203 and the BET bromodomain inhibitor JQ1, which targets MYC transcription, synergistically suppressed MYC expression and cell survival in vitro as well as tumor xenograft growth. Furthermore, MUC1 expression significantly correlated with that of MYC and its target genes in human KRAS mutant NSCLC tumors. Taken together, these findings suggest a therapeutic approach for targeting MYC-dependent cancers and provide the framework for the ongoing clinical studies addressing the efficacy of MUC1-C inhibition in solid tumors., (©2016 American Association for Cancer Research.)

MUC1 is overexpressed in human carcinomas. The transcription factor SNAIL can activate epithelial-mesenchymal transition (EMT) in cancer cells. In this study, in renal carcinoma, we demonstrate that (i) MUC1 and SNAIL were overexpressed in human sarcomatoid carcinomas, (ii) SNAIL increased indirectly MUC1 expression, (iii) MUC1 overexpression induced EMT, (iv) MUC1 C-terminal domain (MUC1-C) and β-catenin increased SNAIL transcriptional activity by interaction with its promoter and (v) blocking MUC1-C nuclear localization decreased Wnt/β-catenin signaling pathway activation and SNAIL expression. Altogether, our findings demonstrate that MUC1 is an actor in EMT and appears as a new therapeutic target., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

MUC1 is a membrane-anchored mucin and its cytoplasmic tail (CT) can interact with many signaling pathways and act as a co-transcription factor to activate genes involved in tumor progression and metastasis. MUC1 is overexpressed in renal cell carcinoma with correlation to prognosis and has been implicated in the hypoxic pathway, the main renal carcinogenetic pathway. In this context, we assessed the effects of MUC1 overexpression on renal cancer cells properties. Using shRNA strategy and/or different MUC1 constructs, we found that MUC1-extracellular domain and MUC1-CT are involved in increase of migration, cell viability, resistance to anoikis and in decrease of cell aggregation in cancer cells. Invasiveness depends only on MUC1-CT. Then, by using siRNA strategy and/or pharmacological inhibitors or peptides, we showed that sheddases ADAM10, ADAM17 and gamma-secretase are necessary for MUC1 C-terminal subunit (MUC1-C) nuclear location and in increase of invasion property. Finally, MUC1 overexpression increases ADAM10/17 protein expression suggesting a positive regulatory loop. In conclusion, we report that MUC1 acts in renal cancer progression and MUC1-C nuclear localization drives invasiveness of cancer cells through a sheddase/gamma secretase dependent pathway. MUC1 appears as a therapeutic target by blocking MUC1 cleavage or nuclear translocation by using pharmacological approach and peptide strategies.

Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.

In response to the widespread use of β-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are potent β-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2,6-dimethoxybenzamido)methylboronic acid was identified as being a potent inhibitor of Actinomadura sp. R39 transpeptidase (IC(50): 1.3 μM). In this work, we synthesized and studied the potential of a number of acylaminomethylboronic acids as inhibitors of PBPs from different classes. Several derivatives inhibited PBPs of classes A, B and C from penicillin sensitive strains. The (2-nitrobenzamido)methylboronic acid was identified as a good inhibitor of a class A PBP (PBP1b from Streptococcus pneumoniae, IC(50) = 26 μM), a class B PBP (PBP2xR6 from Streptococcus pneumoniae, IC(50) = 138 μM) and a class C PBP (R39 from Actinomadura sp., IC(50) = 0.6 μM). This work opens new avenues towards the development of molecules that inhibit PBPs, and eventually display bactericidal effects, on distinct bacterial species., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and mimicking the transition state of the enzymatic reaction. We have solved the structures of complexes of a penicillin-binding protein, the DD-peptidase from Actinomadura sp. R39, with four amidomethylboronic acids (2,6-dimethoxybenzamidomethylboronic acid, phenylacetamidomethylboronic acid, 2-chlorobenzamidomethylboronic acid, and 2-nitrobenzamidomethylboronic acid) and the pinacol ester derived from phenylacetamidomethylboronic acid. We found that, in each case, the boron forms a tricovalent adduct with Oγ of Ser49, Ser298, and the terminal amine group of Lys410, three key residues involved in the catalytic mechanism of penicillin-binding proteins. This represents the first tricovalent enzyme-inhibitor adducts observed by crystallography. In two of the five R39-boronate structures, the boronic acid is found as a tricovalent adduct in two monomers of the asymmetric unit and as a monocovalent adduct with the active serine in the two remaining monomers of the asymmetric unit. Formation of the tricovalent complex from a classical monocovalent complex may involve rotation around the Ser49 Cα-Cβ bond to place the boron in a position to interact with Ser298 and Lys410, and a twisting of the side-chain amide such that its carbonyl oxygen is able to hydrogen bond to the oxyanion hole NH of Thr413. Biphasic kinetics were observed in three of the five cases, and details of the reaction between R39 and 2,6-dimethoxybenzamidomethylboronic acid were studied. Observation of biphasic kinetics was not, however, thought to be correlated to formation of tricovalent complexes, assuming that the latter do form in solution. On the basis of the crystallographic and kinetic results, a reaction scheme for this unexpected inhibition by boronic acids is proposed.